1. Field of the Invention
This invention relates to the imbibition, germination and liquid priming of conifer somatic embryos. More particularly, the invention relates to the imbibition, germination and liquid priming of conifer somatic embryos in a manner that provides a scaleable and reproducible means of germinating and facilitating rapid early growth of conifer somatic embryos.
2. Background Art
The development and advancement of somatic embryogenesis as a vegetative propagation technology has made it possible to mass-produce genetically identical individual plants through the asexual reproduction of a source explant (Tautorus at al. 1991, Roberts et al. 1995). This technology can be applied in clonal forestry in plantation programs. The primary advantages of clonal forestry, as defined by Kleinschmit et al. (1993) and Park et al. (1998a), are: 1) the ability to capture a greater portion of the non-additive genetic gain from selected individuals within a breeding population; 2) the capability to rapidly introduce individuals with desirable traits to meet known site conditions; and 3) the ability to carefully plan genetic diversity into plantation programs.
Somatic embryogenesis of woody plants is generally a multi-step process (U.S. Pat. Nos. 4,957,866; 5,183,757; 5,294,549; 5,413,930; 5,464,769; 5,482,857, 5,506,136; the disclosure of all of which are herein incorporated by reference). No matter how diverse the different somatic embryogenesis protocols might be, the one common step is that somatic embryos must be germinated to produce somatic seedlings.
There are two standard approaches for germinating somatic embryos. The first employs conventional in vitro methods and the second uses encapsulation of somatic embryos to produce synthetic seed.
Conventional in vitro methods are generally based on the following steps. Initially, a naked somatic embryo (i.e., an embryo unprotected by any coatings) is sown, using aseptic techniques, onto a sterile semi-solid or liquid medium contained within a solid-support such as a Petri dish or a Phytatray® under sterile conditions, Next, after the somatic embryo has germinated under sterile conditions, the germinant is transplanted into conventional nursery growing systems. Generally, most protocols require that the germinants be autotrophic before they can be sown ex vitro. There are many disadvantages associated with in vitro protocols. The most significant are: 1) the repeated manual handling of each individual embryo in the germination and transplanting steps; 2) the stringent requirement for sterile techniques and culture conditions through all steps until somatic germinants are transplanted out of the in vitro germination environment into horticultural growing media; 3) the length of time (usually several weeks) of in vitro culture required to produce a germinant that is sufficiently robust to survive ex vitro; and 4) the difficulty in acclimatizing in vitro plantlets into ex vitro nursery environments. Therefore, the art of traditional in vitro protocols has an inherent nature of low efficiency and high cost. These characteristics are prohibitive to mass production of somatic seedlings. These undesirable characteristics make the commercial production of somatic seedlings less competitive than that of zygotic seedlings. Automation, including robotics and machine vision, may reduce or eliminate the extensive hand-handling that is currently necessary to germinate naked somatic embryos. However, no commercial equipment currently exists which can reliably, aseptically, and cost-effectively perform the in vitro protocols for germination of naked somatic embryos and subsequent transplanting into conventional propagation systems (Roberts et al., 1995; reviews by Sakamoto et al. 1995).
The second approach utilises encapsulation (generally gel-encapsulation) of the somatic embryos (Carlson and Hartle 1995, Gray et al., 1995; U.S. Pat. Nos. 4,562,663; 4,777,762; 4,957,866; 5,010,685; 5,183,757; 5,236,469; 5,427,593; 5,451,241; 6,486,218; 5,482,857 all of which are herein incorporated by reference) prior to germination. The embryos are encapsulated in various coating materials to form so-called “artificial seed”, “synthetic seed” or “manufactured seed”. This encapsulation process may or may not incorporate nutrients into the encapsulating medium, and provides a means by which the embryos can presumably be sown with conventional nursery seeding equipment (i.e., drum seeders or fluid drill seeders) into conventional nursery growing systems. The prior art makes references to sowing artificial seeds ex vitro into germination media comprised of soil or soil-less mixes, but in fact, the prior art only teaches methods for germinating artificial seeds in vitro, i.e., on sterilised semi-solid laboratory media. It appears that no practical approaches are taught or otherwise disclosed in the prior art for sowing encapsulated somatic embryos and/or artificial seed and/or manufactured seed into conventional growing systems using conventional sowing equipment.
There are also numerous biological and operational disadvantages inherent in using gel-encapsulated somatic embryos. Biologically, the most significant disadvantage is the much lower germination vigour and conversion success into plants than corresponding zygotic seeds, as seen in the prior art protocols for encapsulating or otherwise coating somatic embryos (Redenbaugh et al., 1993; Carlson & Hartle, 1995; Gray et al., 1995). This is in sharp contrast with the germination vigour and conversion success of non-encapsulated or non-coated somatic embryos, produced with methods disclosed in the art, and then sown using aseptic techniques onto in vitro germination media in sterile conditions. The in vitro sown somatic embryos can approximate those of the corresponding zygotic seeds (e.g., greater than 85%) (Gupta and Grob, 1995).
Timmis et al. (U.S. Pat. No. 5,119,588, incorporated herein by reference) recognised that “somatic embryos are too under-developed to survive in a natural soil environment” and therefore must be “cultured with an energy source, such as sucrose”. They identify a method by which plant somatic embryos can be sown into horticultural containers filled with particulate soil-like substrates. Solutions containing compounds serving as carbon and energy sources and other nutrients, such as minerals and vitamins, are added to the substrates before or after the embryos are sown. Because such a “culture medium is highly susceptible to invasion by phytopathogens, which can result in death or retard the growth of the embryos”, they teach that the containers, substrate, nutrient solutions and other components of their system must be biologically sterile. Somatic embryos must be sown into containers using aseptic techniques.
In the past, each sown container had to be kept biologically separated from the others and from the external environment and had to be kept in a sterile condition until the embryo had successfully germinated and developed into a complete, independent autotrophic plant. Only after autotrophy had been reached could the somatic seedlings be removed from the sterile conditions and then transplanted into a conventional commercial propagation environment. Even though the art taught by such methods may be practised to produce somatic seedlings, such methods are labour-intensive and bear characteristics of low efficiency, high cost and impracticability for mass production of somatic seedlings in a nursery environment.
There is a constant need for improvement of these techniques and methods in order to overcome the disadvantages of the germination and growth phases associated with somatic plant embryos. Improvements of high importance are those that would allow for shorter in vitro culture periods, reduced handling, opportunities for automation and the production of germinants or seedlings with high vigour.